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A protocol to quantify spray deposits in grape bunches

Jan-Cor Brink¹, Gustav Holz¹, Frikkie Calitz² & Paul H. Fourie¹*
(1) Department of Plant Pathology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
(2) ARC Biometry Unit, Private Bag X5026, Stellenbosch 7599, South Africa
*email: phfourie@sun.ac.za
Introduction
Various studies revealed that Botrytis cinerea, the causal pathogen of Botrytis bunch rot, is mostly associated with rachises, laterals, pedicels and berry bases, and not with berry skins as previously understood (Holz et al., 2003). Provided that sufficient coverage of inner bunch parts was achieved, laboratory studies have shown that fungicides can effectively reduce the amount of B. cinerea at the various positions in bunches, and prevent infection and symptom expression at all growth stages. The same efficacy was, however, not achieved with the same fungicides when using conventional spraying methods in vineyards (Van Rooi & Holz, 2003). Failure to control Botrytis and other fruit and foliar diseases in vineyards is often attributed to insufficient coverage of susceptible tissue. Research regarding spray application to ensure efficient spray coverage is therefore needed to improve disease management of fruit and foliar diseases in vineyards.

FIG. 1. Mixture of SARDI Yellow Fluorescent Pigment© and the fungicide fenhexamid.

Previously, coverage evaluations in South Africa were based on the use of water-sensitive cards. However this method does not give a good indication of the spray coverage on certain critical positions of the grape bunches. Residue recovery techniques were also used to provide an overall assessment of the quantity of spray deposits, but did not give a good indication of application quality such as uniformity or spray distribution on the leaves and bunch parts (Holownicki et al., 2002). Visual assessment gives an indication of the quality of the application, but the human eye lacks quantitative measuring and speed of measurement. More recently, water-sensitive papers were used for visual assessment and image analyses in spray application experiments (Holownicki et al., 2002). However, to give a true indication of spray deposits and penetration, cards need to be the same size and orientation as the target, and this method does therefore not give a good indication of the spray coverage on the 3-dimensional target sites in bunches. Furthermore, the target to which fungicides are applied changes constantly, because of the transformation of grape bunches during the growth season (Barry & Weber, 2002). Three stages of growth can be distinguished within a season, namely flowering/set, pea-size and bunch closure. Each stage differs in how open or closed the bunch is and therefore influences spray deposition. With an open bunch, for example, increased air velocity blows droplets off the grape berries. In a medium packed bunch, extra air velocity blows drops off the front berries but helps the liquid filter through the bunch to the grapes in the back. In a closely packed bunch, extra blowing makes no difference since filtration is negligible (Barry & Weber, 2002).
As part of a research programme aiming at optimising spray application in vineyards, the Department of Plant Pathology at the University of Stellenbosch developed a spray cover assessment protocol using fluorometry, photomicrography and digital image analyses to measure spray coverage on susceptible grape bunch parts (Brink et al., 2004).

FIG. 2. Bunch parts were illuminated in a hexagonal box fitted with 6 black light tubes (A), visualised under a stereo microscope at 20x magnification (B) and photographed with a digital camera (Nikon DMX 1200) (C).

Spray cover assessment protocol
Bunches are sprayed with a mixture (Fig. 1) of fungicide and Yellow Fluorescent Pigment© (400 g/L, EC) (South Australian Research and Development Institute) at 2 L/100 L at the recommended dose. Sprayed parts from bunches are illuminated under six black lights which are installed in a custom-made hexagonal illumination box (Fig. 2A) that fits closely around a Nikon SMZ 800 stereoscopic zoom microscope (Fig. 2B). Images are digitally captured through a stereoscopic microscope at 20 x magnification using a high-quality photomicrographic digital Nikon DXM 1200 camera (Fig. 2C). Image analysis and enhancements are done with Image-Pro Discovery version 4.5 for Windows (Media Cybernetics) software. In order to reduce background noise (Fig. 3A) and enhance fluorescent pigment, brightness, contrast and gamma settings are adjusted (Fig. 3B). The total areas of deposited pigment in selected areas of interest (AOI) are calculated (Fig. 3C) and the percentage area covered is subsequently calculated for each AOI.
Validation of spray application protocol
Dauphine grape bunches (sampled at pea-size and bunch closure) were sprayed with a mixture of fenhexamid (Teldor© 500 SC, Bayer) at the recommended dose (75 ml/100 L) and Yellow Fluorescent Pigment© (400 g/L, EC) (South Australian Research and Development Institute) at 2 L/100 L (Furness, 2000). Spray volumes ranging from 1 to 15 ml were applied by means of a gravity feed mist spray gun [Fig 4 (ITW DEVILBISS Spray Equipment Products, 195 Internationale Blvd, Glendale Heights IL 60139 USA)] in a spray chamber [660 x 1410 x 800 mm (h/l/w)]. Water sensitive cards (Syngenta SA, Halfway House, 1685) were included in each treatment to visually assess droplet dispersal and dispersal of fluorescent pigment in droplets. Fluorescent pigment coverage data for each spray volume and growth stage were subjected to the appropriate analysis of variance, linear regression analysis and variance component analysis using SAS v 8.2 statistical software.

FIG. 3. Image processing and analysis of a digital taken of a berry skin from a grape bunch that was sprayed with a mixture of fenhexamid and Yellow Fluorescent Pigment©, (A) Selected objects are UV-illuminated and digitally photographed at 20x magnification, (B) subjected to several image contrasting and filtering processes, (C) and AOI selected and the total area of deposited pigment calculated for each AOI using Image-Pro Discovery image analysis software.

FIG. 4. Gravity feed mist spray gun.

Statistical analyses clearly showed that the described protocol could be used to accurately determine coverage on the susceptible bunch parts in grape bunches. Fluorescent pigment coverage had a significant linear fit on spray volume (Fig. 5). An increase in spray volume generally led to an increase in coverage. Coverage was significantly influenced by growth stage and bunch parts. The highest mean fluorescent pigment coverage was measured at pea-size on berry skins, while the lowest mean fluorescent pigment coverage was measured at bunch closure on rachises. In general, pea-size bunches had a higher mean percentage area coverage on the different bunch parts than bunches sprayed at bunch closure. This can be explained by higher porosity of bunches at pea size compared with more compact bunches at bunch closure. Structural bunch parts were furthermore up to three times more difficult to cover than berry skins at both stages. Variance component analysis revealed that variation could be reduced by increasing the number of bunches, rather than the samples per bunch or measurements per image.
Conclusion
Collectively, these results clearly showed that spray applications earlier in the season will result in higher and more effective spray deposition on the susceptible bunch parts. Disease management would thus be most effective since structural bunch parts are most susceptible and pathogen inoculum most abundant during pre-flower to pea-size stages in vineyards (Holz et al., 2003).
The described protocol provides an essential tool that can be used to study the optimisation of spray application of agro-chemicals and/or biological control agents in vineyards. Hence, adequate deposition of active ingredient on the susceptible vegetative and reproductive parts of grapevines for effective pathogen or pest control can be facilitated.
In future studies, minimum coverage levels for effective pathogen control will be determined and subsequently be used as benchmarks to evaluate spray application in vineyards. The technology developed in the Botrytis-grapevine model will directly benefit the management of other foliar and fruit diseases of grapevine, such as powdery and downy mildew as well as diseases or pests in other cropping systems.

FIG. 5. Mean fluorescent pigment coverage (% area) on berry skin, pedicel and rachis (bunch closure stage only) at pea-size and bunch closure stages and linear regression lines fitted on spray volume for part x stage combinations.

Literature cited
Barry, S.I., & Weber, R.O. 2002. The application of pesticides to grape bunches. Pages 28-40 in: In Proceedings of the 2001 Mathematics-in-Industry Study Group, Australia.
Brink, J.C., Holz G., Calitz, F.J., & Fourie, P.H. 2004 Development of a protocol to quantify spray deposits on grape bunches. 7th International Symposium on Adjuvants for Agrochemicals ISAA 2004, ISAA 2004 Foundation, South Africa 230-236.
FURNESS, G.O. 2000. SARDI Fluorescent Pigment suspension concentrate. Fact Sheet 1-2000. South Australian Research and Development Institute, SARDI. SARDI/Primary Industries and Resources.
Holz, G., Gütschow, M., Coertze, S., & Calitz, F.J. 2003. Occurrence of Botrytis cinerea and subsequent disease expression at different positions on leaves and bunches of grape. Plant Disease 87: 351-358.
Holownicki, R., Doruchowski, G., Swiechowski, W., & Jaeken, P. 2002. Methods of evaluation of spray deposit and coverage on artificial targets. Electronic Journal of Polish Agricultural Universities, Agricultural Engineering 5 Issue 1: 1-9.
Van Rooi, C., & Holz, G. 2003. Fungicide efficacy against Botrytis cinerea at different positions on grape shoots. South African Journal of Enology and Viticulture 24: 11-15.

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